New non detrimental DNA binding mutants of the Escherichia coli initiator protein DnaA

Marlene Asklund, Tove Atlung

    Publikation: Bidrag til tidsskriftTidsskriftartikelForskningpeer review

    Resumé

    The initiator protein DnaA has several unique DNA-binding features. It binds with high affinity as a monomer to the nonamer DnaA box. In the ATP form, DnaA binds cooperatively to the low-affinity ATP-DnaA boxes, and to single-stranded DNA in the 13mer region of the origin. We have carried out an extensive mutational analysis of the DNA-binding domain of the Escherichia coli DnaA protein using mutagenic PCR. We analyzed mutants exhibiting more or less partial activity by selecting for complementation of a dnaA(Ts) mutant strain at different expression levels of the new mutant proteins. The selection gave rise to 30 single amino acid substitutions and, including double substitutions, more than 100 mutants functional in initiation of chromosome replication were characterized. The analysis indicated that all regions of the DNA-binding domain are involved in DNA binding, but the most important amino acid residues are located between positions 30 and 80 of the 94 residue domain. Residues where substitutions with non-closely related amino acids have very little effect on protein function are located primarily on the periphery of the 3D structure. By comparison of the effect of substitutions on the activity for initiation of replication with the activity for repression of the mioC promoter, we identified residues that might be involved specifically in the cooperative interaction with ATP-DnaA boxes.
    OriginalsprogEngelsk
    TidsskriftJournal of Molecular Biology
    Vol/bind345
    Udgave nummer4
    Sider (fra-til)717-730
    ISSN0022-2836
    StatusUdgivet - 2004

    Citer dette

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    title = "New non detrimental DNA binding mutants of the Escherichia coli initiator protein DnaA",
    abstract = "The initiator protein DnaA has several unique DNA-binding features. It binds with high affinity as a monomer to the nonamer DnaA box. In the ATP form, DnaA binds cooperatively to the low-affinity ATP-DnaA boxes, and to single-stranded DNA in the 13mer region of the origin. We have carried out an extensive mutational analysis of the DNA-binding domain of the Escherichia coli DnaA protein using mutagenic PCR. We analyzed mutants exhibiting more or less partial activity by selecting for complementation of a dnaA(Ts) mutant strain at different expression levels of the new mutant proteins. The selection gave rise to 30 single amino acid substitutions and, including double substitutions, more than 100 mutants functional in initiation of chromosome replication were characterized. The analysis indicated that all regions of the DNA-binding domain are involved in DNA binding, but the most important amino acid residues are located between positions 30 and 80 of the 94 residue domain. Residues where substitutions with non-closely related amino acids have very little effect on protein function are located primarily on the periphery of the 3D structure. By comparison of the effect of substitutions on the activity for initiation of replication with the activity for repression of the mioC promoter, we identified residues that might be involved specifically in the cooperative interaction with ATP-DnaA boxes.",
    keywords = "mutagenic PCR, dnaA(Ts) complementation, mioC promoter regulation, DNA binding motifs, ATP-DnaA",
    author = "Marlene Asklund and Tove Atlung",
    year = "2004",
    language = "English",
    volume = "345",
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    journal = "Journal of Molecular Biology",
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    New non detrimental DNA binding mutants of the Escherichia coli initiator protein DnaA. / Asklund, Marlene; Atlung, Tove.

    I: Journal of Molecular Biology, Bind 345, Nr. 4, 2004, s. 717-730.

    Publikation: Bidrag til tidsskriftTidsskriftartikelForskningpeer review

    TY - JOUR

    T1 - New non detrimental DNA binding mutants of the Escherichia coli initiator protein DnaA

    AU - Asklund, Marlene

    AU - Atlung, Tove

    PY - 2004

    Y1 - 2004

    N2 - The initiator protein DnaA has several unique DNA-binding features. It binds with high affinity as a monomer to the nonamer DnaA box. In the ATP form, DnaA binds cooperatively to the low-affinity ATP-DnaA boxes, and to single-stranded DNA in the 13mer region of the origin. We have carried out an extensive mutational analysis of the DNA-binding domain of the Escherichia coli DnaA protein using mutagenic PCR. We analyzed mutants exhibiting more or less partial activity by selecting for complementation of a dnaA(Ts) mutant strain at different expression levels of the new mutant proteins. The selection gave rise to 30 single amino acid substitutions and, including double substitutions, more than 100 mutants functional in initiation of chromosome replication were characterized. The analysis indicated that all regions of the DNA-binding domain are involved in DNA binding, but the most important amino acid residues are located between positions 30 and 80 of the 94 residue domain. Residues where substitutions with non-closely related amino acids have very little effect on protein function are located primarily on the periphery of the 3D structure. By comparison of the effect of substitutions on the activity for initiation of replication with the activity for repression of the mioC promoter, we identified residues that might be involved specifically in the cooperative interaction with ATP-DnaA boxes.

    AB - The initiator protein DnaA has several unique DNA-binding features. It binds with high affinity as a monomer to the nonamer DnaA box. In the ATP form, DnaA binds cooperatively to the low-affinity ATP-DnaA boxes, and to single-stranded DNA in the 13mer region of the origin. We have carried out an extensive mutational analysis of the DNA-binding domain of the Escherichia coli DnaA protein using mutagenic PCR. We analyzed mutants exhibiting more or less partial activity by selecting for complementation of a dnaA(Ts) mutant strain at different expression levels of the new mutant proteins. The selection gave rise to 30 single amino acid substitutions and, including double substitutions, more than 100 mutants functional in initiation of chromosome replication were characterized. The analysis indicated that all regions of the DNA-binding domain are involved in DNA binding, but the most important amino acid residues are located between positions 30 and 80 of the 94 residue domain. Residues where substitutions with non-closely related amino acids have very little effect on protein function are located primarily on the periphery of the 3D structure. By comparison of the effect of substitutions on the activity for initiation of replication with the activity for repression of the mioC promoter, we identified residues that might be involved specifically in the cooperative interaction with ATP-DnaA boxes.

    KW - mutagenic PCR

    KW - dnaA(Ts) complementation

    KW - mioC promoter regulation

    KW - DNA binding motifs

    KW - ATP-DnaA

    M3 - Journal article

    VL - 345

    SP - 717

    EP - 730

    JO - Journal of Molecular Biology

    JF - Journal of Molecular Biology

    SN - 0022-2836

    IS - 4

    ER -